Synthesis of novel 1,8-acridinediones derivatives: Investigation of MDR reversibility on breast cancer cell lines T47D and tamoxifen-resistant T47D.

Multi drug resistance (MDR) is a serious obstacle in the management of breast cancer. Therefore, overcoming MDR using novel anticancer agents is a top priority for medicinal chemists. It was found that dihydropyridines lacking calcium antagonistic activity (e.g acridinediones) possess MDR modifier potency. In this study, the capability of four novel acridine-1,8-diones derivatives 3a-d were evaluated as MDR reversing agents. In addition, the relationship between structural properties and biological effects of synthesized compounds was discussed. In vitro cytotoxicity of acridine-1,8-diones 3a-d derivatives in combination with doxorubicin (DOX) on T47D and tomoxifen-resistant T47D (TAMR-6) breast cancer cell lines were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. Drug resistant index (DRI), which is equal to the ratio of IC50 in drug-resistant cells over IC50 in drug-sensitive cells, was calculated for each substance. Flowcytometry experiments were also implemented to distinguish cells undergoing apoptosis from those undergoing necrosis. The results from MTT and flowcytometry experiments indicated that 1 nM 3c derivative along with DOX significantly (P<0.05) increased the DOX cytotoxicity in T47D and TAMR-6 breast cancer cell lines. Synthesized compounds 3a and 3b also at concentrations of 1 nM with DOX significantly increased the cytotoxicity of DOX on T47D and TAMR-6 breast cancer cell lines. Meanwhile, 3d derivative with DOX did not exhibit good synergistic effect on cytotoxic activity of DOX, and slightly increased DOX cytotoxicity in both cell lines. Our results proposed that 3c may be an attractive lead compound for further development as a chemotherapeutic agent for MDR breast cancer therapy in combination with routine chemotherapeutic agents such as DOX.

Anti-nociceptive and anti-inflammatory effects of cyanocobalamin (vitamin B12) against acute and chronic pain and inflammation in mice.

In this study, the anti-nociceptive and anti-inflammatory effects of cyanocobalamin (Vit B12) against acute and chronic pain and inflammation were evaluated in mice. Vit B12 (0.87, 1 and 1.77 mg/kg) were injected intraperitoneally. The anti-nociceptive effects against acute pain were examined using hot-plate and writhing tests. The chronic pain was examined 14 days after sciatic nerve ligation using the hot-plate test. Morphine (10 mg/kg) was used as a positive control. Anti-inflammatory effects of Vit B12 against acute and chronic inflammation were assessed using xylene-induced edema in ears and granuloma caused by compressed cotton implantation, respectively. In these tests, sodium diclofenac (15 mg/kg) was used as a positive control. Vit B12 showed a dose related effect in acute anti-nociceptive test and increased the anti-nociceptive effect of morphine in chronic treatment. Vit B12 demonstrated an anti-nociceptive effect in chronic studies as single or continues daily treatment and increased significantly the anti-nociceptive effect of morphine. All doses of Vit B12 significantly decreased xylene-induced ear edema. Maximum anti-inflammatory effect (37.5%) was obtained at dose of 1 mg/kg. In chronic inflammation, Vit B12 significantly decreased granuloma formation in mice. In conclusion our work presents some experimental evidence supporting the administration of cyanocobalamin in controlling acute and chronic neuropathic pain. Cyanocobalamin may have anti-inflammatory effect. It may reduce tolerance to anti-nociceptive effect of morphine as well.

Teratogenic effects of HESA-A, a natural anticancer product from Iran, in mice.

HESA-A is a natural product containing herbal and marine animal substances, which has been patented in Iran. It has shown antioxidant, cytotoxic and anticancer effects. The aim of this study was to evaluate the teratogenic effects of HESA-A in mice. HESA-A (50, 100, 200, 400 and 800 mg/kg) was administered orally to pregnant mice on days 6 to 14 of gestation. Mouse reproductive developmental toxicity study was performed according to the ICH guideline. Embryos from the treated dam were sectioned and studied for external morphological abnormalities and skeletal malformations. HESA-A at two doses (400 and 800 mg/kg) slowed weight gain of pregnant mice. These two doses of HESA-A led to reduction in uterus weight (17% and 20% for the 400 and 800 mg/kg doses, respectively), increase in post-implantation resorption (150% and 200%, respectively), reduction in fetus weight (22% and 32%, respectively) and crown-lump length (15% and 19%, respectively). HESA-A at 400 and 800 mg/kg doses caused mild external and skeletal malformation significantly higher than the normal saline group. However, higher doses caused embryo malformations such as short limbs, spinal abnormalities, dermal cysts, microphtalmia and cleft palate. According to this study, only higher doses of HESA-A, which are 20 to 40 times higher than the usual therapeutic doses based on body surface conversion, may cause embryonic toxicity. This provides a reasonable safety margin for the use of HESA-A in pregnancy. Mechanisms of these abnormalities are not clear and need to be determined.

The role of p53 and cell death by apoptosis and necrosis in 4-hydroperoxycyclophosphamide-induced limb malformations.

The exposure of embryonic murine limbs in vitro to an activated analog of cyclophosphamide, 4-hydroperoxycyclophosphamide (4OOH-CPA), induced limb malformations and apoptosis. The purpose of this study was to investigate the role of the tumor suppressor/cell cycle checkpoint gene, p53, and of cell cycle arrest in the response of the limbs to cyclophosphamide. Limbs, excised on day 12 of gestation from wild-type, heterozygous or homozygous p53-knockout transgenic murine embryos, were treated with vehicle (water) or 4OOH-CPA (0.3, 1.0 or 3.0 microgram/ml) and cultured for 6 days. Exposure of wild-type (+/+) limbs to 4OOH-CPA resulted in limb malformations, and reduced limb areas and developmental scores. The homozygous (-/-) limbs were dramatically more sensitive to the effects of 4OOH-CPA, as assessed by limb morphology, area and score. Heterozygous limbs exposed to the drug were intermediate for each parameter. Apoptosis, as assessed by the formation of a DNA ladder, was increased in drug-exposed wild-type limbs, but not in the drug-exposed homozygous limbs. Light and electron microscopy examination of the limbs revealed that drug treatment of wild-type limbs induced the morphological changes typical of apoptosis, particularly in the interdigital regions. In contrast, there was no evidence of apoptosis in homozygous limbs exposed to 4-OOH-CPA; morphological characteristics of necrosis such as cell membrane breakdown, mitochondrial swelling and cellular disintegration were evident throughout these limbs. Heterozygous limbs had cells dying with the characteristics of both apoptosis and necrosis. Fragments of poly(ADP-ribose) polymerase characteristic of necrosis predominated in the drug-treated heterozygous and homozygous limbs. 4-OOH-CPA-treatment of limbs from wild-type embryos led to arrest of the cell cycle at the G1/S phase. No cell cycle arrest was observed after drug treatment of homozygous limbs, in which populations of cells in S and G2/M phases, as well as a population of sub G1 cells, were found. Thus, the presence of p53 and of p53-dependent apoptosis protect organogenesis-stage limbs from insult with a teratogen. The absence of p53 may decrease DNA repair capacity and contribute to the accumulation of DNA damage in limb cells and their daughter cells; the failure of apoptosis to eliminate cells with DNA damage may result in increased cell death by necrosis and major limb malformations.

Transglutaminase and clusterin induction during normal and abnormal limb development in the mouse.

Apoptotic cell death is important in pattern formation in the limb. The purpose of this study was to investigate the relationship between the occurrence of apoptosis in the mouse limb during normal and abnormal development as well as the expression of tissue transglutaminase and clusterin, two proteins associated with apoptotic cell death. Mouse limb buds were cultured in vitro in the absence or presence of a potent teratogen, an activated analog of cyclophosphamide: 4-hydroperoxycyclophosphamide (1 or 10 micrograms/ml). Using whole-mount in situ DNA labeling and confocal microscopy, apoptotic cells were localized in the interdigital areas of control limbs after culture for 24 h. The number of apoptotic cells in the interdigital areas of the limbs was increased in the presence of 4-hydroperoxycyclophosphamide (1 microgram/ml). Exposure to a higher concentration (10 micrograms/ml) of 4-hydroperoxycyclophosphamide further increased the numbers of cells staining positively for apoptosis. The relative abundance of tissue transglutaminase increased 3-4-fold after 6 or 24 h of culture with either concentration of 4-hydroperoxycyclophosphamide; immunoreactive protein in drug-treated limbs decreased to control levels by 48 h. Transglutaminase immunoreactivity was localized in the interdigital areas of limbs 24 h after drug exposure. Clusterin immunoreactivity in the control limbs was weak. The abundance of clusterin was increased 3-4-fold in drug-treated limbs; this induction occurred only after 48 h of culture with 4-hydroperoxycyclophosphamide. Clusterin immunoreactivity in limbs after drug treatment for 48 h was localized to the interdigital areas; immunogold electron microscopy of clusterin expression showed a specific labeling in phagocytosed apoptotic bodies. Thus, the number of cells staining positively for apoptosis in the limb was greatly increased in the interdigital areas during abnormal limb development. The expression of both transglutaminase and clusterin was altered in areas of the limb undergoing apoptosis during abnormal limb development.

Induction of apoptosis and cathepsin D in limbs exposed in vitro to an activated analog of cyclophosphamide.

Apoptosis, a form of active cell death, plays a role during normal limb development. The present study was done to test the hypothesis that the teratogen cyclophosphamide, an alkylating agent and commonly used anticancer drug, produces malformations by disturbing the regulation of apoptosis in the limb. The effects of a preactivated analog of cyclophosphamide, 4-hydroperoxycyclophosphamide, on limb development and on apoptosis in the limb were determined in vitro. Cathepsin D is a lysosomal protease which is induced in tissues undergoing destruction by apoptosis. To further examine the process of apoptosis in the limb, the effects of 4-hydroperoxycyclophosphamide exposure on cathepsin D protein concentration and on the immunolocalization of cathepsin D in limb buds were assessed. Limb buds from gestational day 12 mice were excised and cultured in roller bottles in a chemically defined medium for up to 6 days. The addition of 4-hydroperoxycyclophosphamide (1 or 10 micrograms/ml) to the culture medium produced time- and concentration-dependent limb malformations. Electrophoresis of the DNA extracted from both control and treated limbs revealed a DNA fragmentation pattern characteristic of apoptosis. Limbs cultured in the control medium showed a "DNA ladder" only after 72 hours in vitro; however, those in the drug-treated groups showed fragmentation within 12 hours of drug exposure. Acridine orange staining and examination of cell ultrastructure with the electron microscope further confirmed that apoptotic cell death in the interdigital areas was accelerated in drug-exposed limbs. The relative abundance of cathepsin D in limbs exposed to 4-hydroperoxycyclophosphamide for 24 hours was increased compared to control limbs.(ABSTRACT TRUNCATED AT 250 WORDS)

Massive hyperplastic thymus in a 22-month-old infant.

Huge mediastinal masses in infants are often difficult to diagnose, and thymic enlargement must be considered in each instance, especially when the mass is located anteriorly. In the patient reported on here, the mass occupied approximately 60% of the chest cavity, and surgical intervention was required to establish the precise histological diagnosis. This proved to be massive hyperplastic thymus.

Civilian arterial injuries.

During 1968-1973, 122 patients with 126 arterial injuries were treated. In 94 instances (90 patients), these injuries involved extremities. Systolic blood pressure was below 90 mm Hg upon admission in 55.6% of all patients and 37.7% of those with injuries to arteries of the extremities. The decision for operative exploration and repair of arteries of extremities was based largely on clinical grounds (shock, loss of pulse). Preoperative arteriography was needed infrequently, while operative angiography was nearly routine. Although several cases of late revascularization or traumatic thrombosis of renal artery have been reported, hypertension complicates the postoperative period, and early, aggressive approach is essential. Mortality was 10.6%, from aortic injuries. There were no deaths among patients with arterial injuries distal to inguinal ligament or thoracic outlet. The amputation rate from reconstruction failure was 1.1%, none occurring in the last 3 years of the series. The high patency rate and lack of evidence of pulmonary embolization suggest that associated venous injuries be repaired routinely. Arterial injuries represent ideal lesions (normal arterial wall with excellent run-in and run-off). Prompt treatment of shock and early, proper management of patients' mechanical disruptions will salvage many lives and most limbs.